Deficiency of lysosomal acid alpha glucosidase (GAA) results in glycogen storage diseasae type II (Pompe's disease), encompassing a spectrum of disorders of varying severity ranging from a rapidly fatal infantile onset form to a slowly progressive adult onset form. The infantile form is characterized by massive accumulation of glycogen in cardiac and skeletal muscle and other tissues, while in the adult onset form involvement is limited to skeletal muscle. To study the molecular basis for the clinical heterogeneity of GAA deficiency disease, I sought to clone and analyze the gene for human GAA. I. ISOLATION OF THE GENE To isolate the coding sequences, I screened a lambda gt11 expression library using an affinity purified polyclonal antibody to GAA. Retesting positive phage for reactivity to monoclonal antibodies identified a single phage (containing a 2 Kb cDNA insert). The 2 Kb cDNA hybridized specifically to the portion of chromosome 17 (17q21-23) containing the locus for human GAA and to a 3.4 Kb mRNA, consistent with the size (approx. 105 Kd) of the protein. Finally, the cDNA identified differences in the mRNA of GAA deficients. Thus, in 1 or 2 infantile GAA deficients, the 3.4 Kb mRNA was not detectable, while an adult onset deficient and mRNA of reduced size and amount was found. Based upon the above, I have cloned a 2 Kb cDNA as probe, and have already isolated a longer cDNA (3.4 Kb) 2) determine the complete size of the 5' untranslated region of the cDNA by primer extension and complete sequencing the full length cDNA using a combination of M13 subcloning, deletion subcloning and GAA specific primers. 3) isolate the normal genomic fragment, determine the number of introns and exons, a restriction map, and sequence the splice junctions. II. STUDIES OF DNA AND RNA FROM NUTANT AND GENETICALLY POLYMORPHIC NORMAL CELLS The isolated cDNA or genomic DNA will be used to study mutant cell(s) (A) to determine at the level of DNA the presence of all exons, (B) to determine the presence of normal size and amounts of mRNA and to detect splicing errors and deletions by S1 nuclease analysis. I have also analyzed mRNA from another 11 patients and found the same degree of heterogeneity as in the first three patients. (C) to determine precise mutations including direct cloning and sequencing of mRNA where produced and/or genomic DNA where mRNA is absent. I will initially focus on individuals who express mRNA and who exhibit abnormalities in various properties of the enzyme protein, to provide structure- function relationships.